1. Field of the Invention
The present invention relates to an experimental apparatus for a physiological experiment of a sliced specimen (slice preparation) of biological tissues while maintaining the physiological activities of the sliced specimen of the biological tissue, and a specimen holder (slice holder) to be used in the experimental apparatus.
2. Description of the Related Art
It is a basic technique of the physiological experiments to excise a part of the tissue from a biological specimen and measure its physiological activities while maintaining its physiological activities. Mostly, a sliced specimen excised from the biological tissue (simply abbreviated as a sliced specimen hereinafter), or a thin tissue piece (usually with a thickness of 1 mm or less) excised from the biological specimen is prepared:, and various physiological measurements are carried out using the specimen, because it is necessary to feed oxygen and nutrients over the entire biological tissue and remove waste products in order to maintain physiological activities of the biological tissue.
Two methods have been used for storing the sliced specimen for a long period of time while maintaining its physiological activities. The first method comprises, as shown in FIG. 5, disposing a petri dish 12 with a mesh structure for allowing a solvent to permeate in a vessel 11 filled with a saline solution 1 containing appropriate nutrients, placing a:sliced specimen 13 on the petri dish 12, and immersing and maintaining the sliced specimen 13 in the saline solution 1 while a gas mainly comprising oxygen and carbon dioxide is continuously fed into the saline solution 1 through a gas feed tube 15 equipped with a spherical filter 14.
The second method comprises, as shown in FIG. 6, placing a filter 17 (filter paper or membrane filter) that is permeable to both a solution or gas on a vessel 16 filled with the saline solution 1, mounting a sliced specimen 13 on the filter, housing the overall vessel 16 in a sealed vessel 18, and filling the inside of the sealed vessel 18 with a moist gas generated from the moisturizing saline solution 1.
The apparatuses shown in FIGS. 5 and 6 may be also used for staining the biological specimen with a biological dye. In the apparatus shown in FIG. 5, the sliced specimen 13 is stained by adding a dye in the solution 1 in which the specimen is immersed. In the apparatus shown in FIG. 6, on the other hand, the sliced specimen 13 is stained by dropping a staining solution on the specimen.
While both of the methods described above have almost the same effect for maintaining the sliced specimen 13 for a long period of time, it is a problem that the physiological activity of the sliced specimen 13 has been decreased because the sliced specimen 13 tends to be deformed and damaged when the sliced specimen 13 is transferred to a different site by taking the specimen out of the vessels 11 and 18.
While the sliced specimen 13 is transferred by sucking it with a pipette together with the solution 1 in the first method, deformation of the thin and soft sliced specimen 13 is inevitable, sometimes dealing a fatal damage to the sliced specimen 13.
While the sliced specimen 13 is peeled off from the filter 17 to transfer it into the solution in a separate vessel, the sliced specimen 13 is also liable to be imparted with a fatal damage during the transfer process. Since the sliced specimen 13 stored for a long period of time is often adhered onto the filter 17, a special care is required for peeling the sliced specimen 13 from the filter 17.
A high concentration of the dye should be maintained for a long period of time around the sliced specimen 13 in order to sufficiently and uniformly stain the biological specimen. Accordingly, the dye should be distributed in the overall solution 1 dipping the sliced specimen 13 when the apparatus shown in FIG. 5 is used for staining, which is very uneconomical because a large quantity of the usually expensive dye is needed. While consumption of the dye may be saved in the apparatus shown in FIG. 6, staining tends to be poor since it is difficult to allow a sufficient amount of the staining solution to stay around the sliced specimen 13.
Properly introducing the sliced specimen 13 into a measuring apparatus And holding the specimen there is important for measuring the physiological activities of the sliced specimen 13. The sliced specimen 13 is introduced into the measuring apparatus using a pipette together with the solution, as has been described previously. However, the physiological activities of the sliced specimen 13 may be possibly compromised by the impact of the transfer operation or by environmental changes.
It is also necessary to immerse the sliced specimen 13 in the saline solution 1, and the solution around the sliced specimen 13 should be steadily circulated in the measuring apparatus. While an appropriate weight is used for fixing the sliced specimen 13 in order to avoid the sliced specimen 13 from being displaced by the stream of the solution, the physiological activities of the sliced specimen 13 may be compromised by stress caused by the weight. The weight may also interfere the physiological measurements such as observation of transmission light and electrical potential measurements, and imaging using a dye.
The apparatus shown in FIG. 6 may be used for a cultivator by maintaining the inside of the sealed vessel 18 aseptic. However, it is difficult for the sliced specimen 13 to be transferred into the measuring apparatus without being damaged after peeling off the sliced specimen 13 from the filter 17, because the sliced specimen 13 is adhered on the filter 17. It is also impossible in general to withdraw the sliced specimen 13, subject to a aseptic treatment and resume culturing by putting it back to the cultivator after the measurement without dealing a damage to the specimen.
Accordingly, it is an object of the present invention devised for solving the technical drawbacks of the conventional art to provide an experimental apparatus for a sliced specimen of biological tissues and a specimen holder, wherein the conditions required for preservation, staining, physiological measurements and cultivation are satisfied while maintaining the sliced specimen in a condition suitable for maintaining physiological activities in each conditions above, besides subjecting the sliced specimen to an experiment by readily permitting the specimen to be transferred without being physically damaged.
A first aspect of the present invention provides an experimental apparatus for a sliced specimen of a biological tissue that is used for a physiological experiment by holding the sliced specimen of the biological tissue in a circulating saline solution while maintaining its physiological activities, comprising an experimental vessel for housing the saline solution, a specimen holder for holding the sliced specimen of the biological tissue, and a device for holding the holder for allowing the specimen holder to be attached to and detached from a prescribed position in the experimental vessel.
A second aspect of the present invention provides the specimen holder comprises an annular holder frame and a membrane filter for closing the lower part of the holder frame, the sliced specimen of the biological tissue being held on the membrane filter, in the experimental apparatus for a sliced specimen of a biological tissue of the first aspect.
A third aspect of the present invention provides holder frame has a height by ⅕ or less as small as its inner diameter, in the experimental apparatus for a sliced specimen of a biological tissue of the second aspect.
A fourth aspect of the present invention provides the membrane filter is made of a material on which the sliced specimen of the biological tissue can adheres, in the experimental apparatus for a sliced specimen of a biological tissue of the second aspect or the third aspect.
A fifth aspect of the present invention provides the membrane filter is made of a material that enhances its transparency by moisturizing, in the experimental apparatus for a sliced specimen of a biological tissue of any one of the second aspect to the fourth aspect.
A sixth aspect of the present invention provides the membrane filter has a good permeability to a gas and solution required for maintaining physiological conditions of the sliced specimen of the biological tissue, in the experimental apparatus for a sliced specimen of a biological tissue of any one of the second aspect to the fifth aspect.
A seventh aspect of the present invention provides the portion at the bottom of the experimental vessel located below the holding position of the specimen holder by the holder holding device comprises a transparent member, in the experimental apparatus for a sliced specimen of a biological tissue of any one of the first aspect to the sixth aspect.
Also, an eighth aspect of the present invention provides a specimen holder constructed so as to be able to be held by being fixed at a prescribed site in the experimental vessel by attaching to the holder holding device of the experimental apparatus for the sliced specimen of the biological tissue of the first aspect, and so as to be able to attach to and detach from the holder holding device, comprising an annular holder frame to be held by the holder holding device and a membrane filter for closing the bottom part of the holder frame, the sliced specimen of the biological tissue being held on the membrane filter.
A ninth aspect of the present invention provides the holder frame has a height by ⅕ or less as small as its inner diameter in the specimen holder in the eighth aspect.
A tenth aspect of the present invention provides the membrane filter is made of a material on which the sliced specimen of the biological tissue can adheres in the specimen holder of the eighth aspect or the ninth aspect.
An eleventh aspect of the present invention provides a material that enhances its transparency by moisturizing is used for the material for the membrane filter in the specimen holder of any one of the eighth aspect to the tenth aspect.
A twelfth aspect of the present invention provides the membrane filter has a good permeability to a gas and solution required for maintaining physiological conditions of the sliced specimen of the biological tissue in the specimen holder of any one of the eighth aspect to the eleventh aspect.
The first aspect of the present invention so configured as described above enables an experiment comprising the steps of introducing the sliced specimen of the biological tissue with the specimen holder into the experimental vessel in which the saline solution is circulating, readily disposing the sliced specimen of the biological tissue at a prescribed site in the experimental vessel by attaching the specimen holder to the holder holding device, and readily taking the sliced specimen of the biological tissue out of the experimental vessel together with the specimen holder by detaching the specimen holder from the holder holding device after completing the experiment. Since the sliced specimen of the biological tissue is transferred together with the specimen holder, the sliced specimen of the biological tissue can be readily transferred without dealing a physical damage to the specimen. Consequently, the sliced specimen of the biological tissue can be withdrawn together with the specimen holder after completing the experiment, and subjected to an septic treatment to resume cultivation by returning the intact specimen to the cultivator.
According to the second aspect and the eighth aspect of the present invention, the sliced specimen of the biological tissue is held while maintaining its physiological activities besides permitting the sliced specimen of the biological tissue to be readily transferred without dealing a physical damage to the specimen, using a specimen holder having a simple structure comprising: an annular holder frame and a membrane filter for closing the lower part of the frame folder. Since good staining of the sliced specimen of the biological tissue is possible using a small quantity of a staining solution by allowing an amount of the staining solution sufficient for staining the sliced specimen to pool around the sliced specimen by injecting the staining solution to the inside of the holder frame of the specimen holder holding the sliced specimen of the biological tissue, the expense of the experiment can be saved by largely reducing consumption of the staining solution.
According to the third aspect and the ninth aspect of the present invention, the experimental work such as attaching electrodes to the sliced specimen of the biological tissue while holding the sliced specimen of the biological tissue with the specimen holder is made easy since the holder frame of the specimen holder has a height by ⅕ as small as the inner diameter of the holder.
According to the fourth aspect and the tenth aspect of the present invention, displacement of the sliced. specimen of the biological tissue when the sliced specimen of the biological tissue is transferred together with the specimen holder or the solution around the specimen is exchanged, and by circulation of the solution in the experimental vessel, can be prevented, since the sliced specimen of the biological tissue is adhered to and integrated with the membrane filter on the specimen holder. Accordingly, a weight for pressing the sliced specimen of the biological tissue is not needed because the sliced specimen of the biological tissue is not displaced by the flow of the solution during the experiment. Consequently, drawbacks that the physiological activities of the sliced specimen of the biological tissue are compromised by being pressed with the weight can be solved, besides eliminating interference of the weight for physiological measurements such as observation of transmission light, measurement of electrical potential and imaging using a dye.
According to the fifth aspect and the eleventh aspect of the present invention, experiments and observation of the sliced specimen of the biological tissue are possible while mounting the specimen on the specimen holder, because the membrane filter turns out to be more transparent when the specimen holder is dipped in the saline solution.
According to the sixth aspect and the twelfth aspect of the present invention, physiological state of the sliced specimen of the biological tissue can be maintained in good conditions during the experiments.
According to the seventh aspect of the present invention, observation by a transmission light is possible by attaching the specimen holder holding the sliced specimen of the biological tissue to the holder holding device and by illuminating the specimen with an illumination light from below the experimental vessel, since good physiological conditions of the sliced specimen of the biological tissue are maintained by circulating the saline solution in the experimental vessel.